Epigenetic regulation of gene expression via methylation has been implicated in diverse diseases including cancer, diabetes and inflammation, and high throughput screening for histone methyltransferase (HMT) inhibitors is an area of intense drug discovery effort. However, there are significant shortcomings with existing HMT enzyme assay methods, and these are slowing exploration of the therapeutic potential of these emerging targets. Detection of specific methylation events can be quite complicated, and detection of S-adenosylhomocysteine (SAH), the invariant product of all HMT reactions, would be preferred in most cases. However, HMTs are very poor catalysts and many have very low SAM requirements - a combination of factors that creates very stringent sensitivity requirements for SAH-based assay methods. Moreover, direct detection of SAH is a very challenging molecular recognition problem as it requires a reagent capable of discriminating between SAH and S- adenosylmethionine (SAM), which differ by a single methyl group. The available SAH assays - which rely on enzymatic conversion of SAH to a detectable product - are inherently prone to interference from screening compounds and lack the sensitivity needed for detection of some methyltransferases. To overcome this technical gap, we propose to leverage the exquisite selectivity and affinity of naturally occurring SAH-binding RNA aptamers, or riboswitches, that control the expression of SAM recycling genes in bacteria. This is a collaborative effort between Dr. Ronald Breaker, who discovered riboswitches in 2002, and BellBrook Labs. Dr. Breaker will use a bioinformatics approach to identify candidate SAH riboswitches with suitable properties from more than 1,000 that are known and characterize the SAH/SAM binding properties of the most promising candidates. BellBrook will incorporate these into a fluorescent SAH sensor suitable for high-throughput screening (HTS) assays and validate it for detection of purified HMTs. This will be the first commercial HTS assay based on an aptamer, and it will overcome the very challenging SAH/SAM discrimination problem with at least 10- fold greater selectivity than has been possible with antibodies. By enabling direct, highly sensitive detection of SAH, the SAH riboswitch sensor (rSen-SAH), will accelerate the screening and profiling of otherwise intractable methyltransferase targets, and thereby make an important contribution to the promising field of epigenetic drug discovery.